Controlled nucleation and growth of hydroxyapatite (HA) crystals on electrospun fibers should play important roles in fabrication of composite scaffolds for bone tissue engineering, but no attempt has been made to clarify the effects of chemical group densities and the cooperation of two and more groups on the biomineralization process. The aim of the current study was to investigate into HA nucleation and growth on electrospun poly(dl-lactide) fibers functionalized with carboxyl, hydroxyl and amino groups and their combinations. Electrospun fibers with higher densities of carboxyl groups, combination of hydroxyl and carboxyl groups with the ratio of 3/7, and combination of amino, hydroxyl and carboxyl groups with the ratio of 2/3/5 were favorable for HA nucleation and growth, resulting in higher content and lower crystal size of formed HA. Carboxyl groups were initially combined with calcium ions through electrostatic attraction, and the introduction of hydroxyl groups could modulate the distance between carboxyl groups. The introduction of amino groups may lead to the inner ionic bonding with carboxyl groups, but can accelerate phosphate ions to form HA through a chelate ring with the calcium ion and carbonyl oxygen. The biological evaluation indicated that the mineralized scaffolds acted as an excellent cell support to maintain desirable cell-substrate interactions, to provide favorable conditions for cell proliferation and to stimulate the osteogenic differentiation.